The present invention is connected to the chemical synthesis of ribonucleic acids (ribo-oligonucleotides, RNA), especially to the machine synthesis of such oligomers as well as to the synthesis of structurally related derivatives of such oligomers.
Pure oligonucleotides of a defined sequence prepared in a chemical synthesis are e.g. used in the field of structural analysis of unit crystals by means of X-ray diffraction or by means of nuclear magnetic spectroscopy. This kind of research contributes to the understanding of biological processes on a molecular level and among other things makes, development of novel therapy concepts possible. Ribonucleic acids constituting a central biological class of compounds (messenger-RNA, transfer-RNA, ribosomal-RNA) are interesting objects for medical chemistry. In this context the availability of assays for fast and reliable testing of compounds potentially interacting with RNA is highly desirable. By chemical synthesis (opposed to production using enzymes, organisms etc.) of such oligonucleotides for testing, introduction of purposeful modifications becomes possible which modifications e.g. allow simple quantification of a desired interaction or make a specific interaction accessible to precise examination.
Natural and modified RNA-oligonucleotides also find use as tools for selective recognition and/or selective modification of RNA- and DNA-oligonucleotide-sequences and other compounds (aptamers and ribozymes). Improvements to be achieved in the chemical synthesis of such compounds could make the introduction of purposeful modifications possible and thus considerably extend the field of application of the types of compounds as described above in medical diagnostics and therapy.
All known methods for chemical synthesis of RNA-oligonucleotides and derivatives thereof are related to concepts which have been very successfully developed for the synthesis of DNA-oligonucleotides (2'-desoxyribonucleic acids, opposed to RNA which comprises a hydroxy-group in the 2'-position). The machine synthesis of DNA- and RNA-oligonucleotides is normally based on a protected nucleoside-derivative immobilized on a solid phase to which further protected nucleoside-derivatives are coupled in steps of one synthesis cycle each until the desired length of chain is achieved. Finally the built-up sequence is freed of all protection-groups and separated from the solid phase.
Ribonucleoside-derivatives for application in the chemical synthesis of ribonucleic acids comprise a D- or L-ribose-unit and have the following general structural formula: ##STR2## whereby R.sup.1 is a base of the purine- or pyrimidine-family or a derivative of such a base,
R.sup.2 is a proton or a substituted derivative of phosphonic acid, PA1 R.sup.3 is a proton or a protection-group for the oxygen atom in 5'-position, PA1 X is a protection-group for the oxygen atom in 2'-position. PA1 The introduction of the protection-group has to be as simple as possible and has to yield uniform compounds which are as free from isomerization products as possible. PA1 The protection-group has to be absolutely stable under the coupling conditions. PA1 The protection-group has to have a structure which allows high coupling yields. PA1 The protection-group has to be completely removable without decomposition or chemical change of the compound to be prepared.
The protection-group X for the oxygen atom in 2'-position is to fulfil substantially the following conditions:
The protection-groups which have been used so far for the 2'-position substantially belong to the three following types. A number of further such protection-group types exist. However, they have not been completely successful for different reasons.
a) Acid-sensitive 2'-O-acetal-protection-groups [1]:
EXAMPLE: X= ##STR3##
This type of protection-group is easily introduced and the chemicals required for establishing such protection-groups as well as the nucleosides carrying them are commercially available. Disadvantages of the protection type are the facts that the protection-groups are not completely stable on synthesis of the chains, that using such protected nucleoside-derivatives only moderate coupling yields are achievable and that isomerization on de-protection is possible.
b) Photo-sensitive 2'-O-ortho-nitrobenzyloxymethyl-protection-groups [2]:
Example: X= ##STR4##
This type of protection-group is easily introduceable, de-protecting is completely orthogonal and good coupling yields are achievable. Disadvantages of this type of protection-group are the facts that complete de-protecting is sometimes not possible and that the chemicals necessary for establishing the protection-groups are not available on the market.
c) Fluoride-sensitive 2'-O-silyl-protection-groups [3]:
Example: X= ##STR5##
This type of protection-group is easily and completely removable (de-protecting), the application is established and the necessary chemicals are available on the market. Disadvantages of this type of protection-groups are the facts that they are difficult to be introduced, that the ribonucleoside-derivatives prepared by introducing the protection-group contain isomers and that only moderate coupling yields are achievable.
The object of the invention is to create ribonucleoside-derivatives comprising a protection-group in 2'-O-position, whereby the inventive nucleoside-derivatives unite the advantages of nucleoside-derivatives with known protection-groups as mentioned above under b) and c) (good introduceability of the protection-group, good stability of the protection-group, high coupling yields and simple de-protecting) but do not show the known disadvantages, in particular no isomerization on introducing and on removing the protection-group.
The use of the inventive ribonucleoside-derivatives protected with a 2-O-protection-group are to allow a chemical synthesis of oligo-ribonucleic acids and of structurally related derivatives thereof with a substantially higher yield and with higher purity than is possible with known units of this kind and therefore, are to make synthesis of substantially longer chains possible.
Furthermore, it is the object of the invention to provide a method for preparing the inventive ribonucleoside-derivatives.